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Abstract

Background

Milk protein fractions and peptides have been shown to have bioactive properties.
This preliminary study examined the potential mechanisms of action and clinical safety
of novel milk protein peptide (MP).

Findings

A novel MP mixture inhibits the tyrosine kinase activity of epidermal growth factor
receptor (EGFR), vascular endothelial growth factor receptor 2 (VEGFR2), and insulin
receptor (IR) with IC50 of 9.85 μM, 7.7 μM, and 6.18 μM respectively. In vitro, this multi-kinase inhibitor causes apoptosis in HT-29 colon cancer cells, and in
a C. elegans worm study, showed a weak but significant increase in lifespan. A six week double-blind,
placebo-controlled study involving 73 healthy volunteers demonstrated that the MP
mixture is safe to consume orally. All clinical blood markers remained within normal
levels and no clinically significant side effects were reported. There was some evidence
of improved insulin sensitivity, neutrophil-to-lymphocyte ratio (NLR), and quality
of life assessment of role of physical function.

Conclusions

These data in combination with the observed in vitro anti-cancer properties warrant further clinical studies to investigate this MP mixture
as a potential clinical nutrition intervention for improving the quality of life and
clinical outcomes in cancer patients.

Trial Registration

Keywords:

Findings

Clinical nutrition products are specifically formulated nutrients to help people manage
various health conditions. With recent advances in biotechnology, new techniques and
tools are now available to isolate components from food that have additional health
enhancing properties. Peptides are good candidates for advanced clinical nutrition
and supplements since they are easily absorbed, and unlike many plant based compounds,
peptides are amino acids that can be eliminated naturally by the body with less potential
toxicity [1]. We have isolated a peptide mixture from regular cow's milk that preliminary basic
and clinical research indicates may have some beneficial bioactive properties. The
purpose of this study was to: (1) characterize the molecular mechanism of action;
(2) assess the mixture's safety in healthy human subjects; and, (3) look for trends
in potential improvements that can be further explored in future clinical trials in
cancer patients.

Methods

Milk peptides

A milk peptide product AminoAct® (MP) was prepared from normal cow's milk and supplied by OncoNutrition (Riverside, CA). A whey based peptide fraction was isolated from pasteurized skim milk using proprietary
enzyme digestion and buffers. The final product was supplied in an aqueous solution.
Table 1 presents the nutrient panel of the MP product examined in this report.

HT-29 Colon Cancer Cell Apoptosis Assay

HT-29 human colon cancer cells were grown in a 72-well Terasaki plate until 80% confluent
in McCoy's 5A medium, 10% FBS [2]. Vehicle, commercial hydrolyzed whey protein (Thorne Research, Sandpoint, ID), and MP mixture were added separately to each well in triplicates for a final concentration
of 0.68 μg/μl media. Pictures were taken after 20 hr incubation.

C.elegans Lifespan Assay

The effects of the MP mixture on the lifespan of C. elegans were performed at the University of Utah C. elegans Research Core Facility (Salt Lake City, Utah) using previously published methods [3]. Nematode Growth Media (NGM) agar plates were prepared containing either no peptides,
or 20 μg/ml, 6.66 μg/ml, 0.66 μg/ml concentrations of the MP mixture. All plates were
seeded with E. Coli strain OP50. Experimental plates were prepared in triplicate. Approximately 10 N2
(wild type strain) worms were cultured on four standard agar plates or plates containing
different dosages of MP. In addition to using N2 worms on standard plates as controls,
other control plates included 40L4 mutant daf-2 (el370) and 40 L4 daf-16(m26) larvae.
The mean number of surviving worms for each day was recorded, and the number of animals
died on each day was calculated and the values were used in log rank tests to examine
statistically significant differences. The results were simultaneously evaluated using
the χ2 test and values above 1.0 at a p-value of > 0.01 are considered statistically different
when comparing longevity of N2 worms on standard plates versus the test sample.

Clinical Safety Study

Subjects

The clinical trial was conducted as a placebo-controlled, double-blind, parallel arm
repeated measures study conducted in a university setting. The protocol was reviewed
and approved by a human participants internal review board (IRB) and written informed
consent was obtained from all patients prior to the initiation of study. Subjects
who were apparently healthy between the ages of 35 - 60 years who had body mass index
(BMI) ranging between 27 and 40 with no known metabolic disorders, history of milk
allergies, and/or medical conditions were eligible to participate in the study. Subjects
were matched according to gender, age, and body mass and randomized to consume a placebo
or the MP mixture in a free living condition. Table 2 presents participant demographics based on gender and group randomization. Eighty-one
subjects volunteered to participate in this study with 73 completing the study. Two
males and six females did not complete the study. Three cited time constraints with
their schedules, two missed multiple testing sessions, 2 did not give a specific explanation
and one participant who was in the supplement group withdrew due to gastrointestinal
distress which could have been instigated by ingestion of the milk or supplement.

Supplementation protocol

The MP mixture or a flavored water and glycerol placebo were administered in three
escalating dosages based on body weight over a six week period according to the supplementation
schedule described in Table 3. Subjects took 1/2 dosage, mixed with 1/2 cup milk immediately after breakfast and
consumed the remaining 1/2 dosage with 1/2 cup milk immediately after dinner. Supplementation
compliance was monitored by having participants return empty bottles of the supplements
as well as submitting a supplement log documenting when supplements were ingested.

Procedures

Participants recorded all food and fluid intake for four days prior to each testing
session which included three weekdays and one weekend day. Dietary inventories were
reviewed by a registered dietitian and analyzed using the ESHA Food Processor (Version 8.6) Nutritional Analysis software (ESHA Research Inc., Salem, OR). Height and body mass were determined using a calibrated electronic scale (Cardinal Detecto Scale Model 8430, Webb City, Missouri). Body composition (excluding cranium) was assessed using a Hologic Discovery W (Hologic Inc., Waltham, MA) dual energy x-ray absorptiometer (DXA) equipped with APEX Software (APEX Corporation Software, Pittsburg, PA). Resting heart rate was determined by palpitation of the radial artery and blood
pressure was assessed by auscultation of the brachial artery using a mercurial sphygmomanometer
using standard clinical procedures [4].

Whole blood samples were analyzed for complete blood counts with platelet differentials
using an Abbott Cell Dyn 3500 (Abbott Laboratories, Abbott Park, IL) automated hematology analyzer. Serum samples were analyzed for a complete metabolic
panel using a Dade Behring Dimension RXL (Siemans AG, Munich, Germany) automated clinical chemistry analyzer. Coefficient of variation (range: 1.0 to 9.6%)
for tests performed was similar to previously published data for these tests [5]. Serum insulin and leptin were determined using commercially available immuno-absorbent
assay (ELISA) kits (Diagnostic Systems Laboratories, Webster, TX) using a Wallac Victor-1420 microplate reader (Perkin-Elmer Life Sciences, Boston, MA) according to kit specifications. Intra-assay and inter-assay coefficient of variation
were 4%-7% for insulin and 2% - 8% for leptin. The homeostasis model assessment for
estimating insulin resistance (HOMAIR) was calculated as the product of fasting glucose times fasting insulin expressed
in conventional units divided by 405 [6]. Participants also completed the SF-36 Quality of Life (QOL) inventory which has
been used to assess physical and mental components related to QOL [7] as well as a symptom and side effect questionnaire.

Statistical analysis

Related variables were grouped together and analyzed by multivariate analysis of variance
(MANOVA) with repeated measures using PASW Statistics 18.0.2 software (SPSS, Chicago, IL). Non-correlated variables were analyzed by univariate repeated measures analysis
of variance (ANOVA). Delta values were calculated and analyzed on select variables
by MANOVA for repeated measures to assess changes from baseline values. Data were
considered statistically significant when the probability of type I error was 0.05
or less. If a significant group, treatment and/or interaction alpha was observed,
Tukey's honestly significant differences post-hoc analyses were performed.

Results

Basic Research Findings

A proprietary MP mixture was screened for tyrosine kinase inhibitory activity. Notably,
this particular peptide mixture's IC50 for EGFR, VEGFR2, and IR are 9.85 μM, 7.7 μM, and 6.18 μM respectively (Figure 1). In vitro, the peptide mixture at 0.68 μg/μl causes cell death in HT-29 human colon cancer
cell line; whereas commercial hydrolyzed whey powder did not cause cell death (Figure
2). Hoechst dye staining showed the cell death is accompanied with nuclear condensation
and DNA fragmentation, suggesting an apoptotic cell death, and the apoptotic death
is dose dependent (data not shown). In vivo, C.elegans worms were used to test the impact of MP on lifespan. Wild type L4 larval N2 worms
were fed with or without the MP. In the group that was fed with agar containing 20
μg/ml peptides, the median survival where 50% worms were alive was Day 15, compared
to Day 13. The last worm in the controlled group died at Day 20, in contrast to Day
25 in the experimental group fed with agar containing milk peptides (Figure 3). Statistical analysis of N2 standard vs.MP fed worms showed a value of 6.1 (p =
0.014) from χ2 test, indicating a weak but significant statistical significance. Lower concentrations
of peptides did not yield significant lifespan expansion (data not shown).

MANOVA analysis indicated that a group × time trend (p = 0.06) was observed between
groups in QOL indices. As Figure 6 presents, univariate ANOVA analysis revealed that participants in the MP group observed
a significantly greater increase (p = 0.01) in role physical (i.e., ability to work
and perform daily activities). No significant side effects were observed for frequency
of dizziness, racing heart rate, skip heart beats, shortness of breath, nervousness,
or blurred vision. Moreover, no subject was medically removed from the study or was
referred for medical consultation.

Figure 6.Comparison between placebo and MP group in role physical measure of quality of life.

Discussion

Milk represents a unique source of nutrients and biologically active components that
act in synergy, as well as independently. Emerging evidence indicates that the protein
component of milk represents a variety of biologically active proteins/peptides that
function as anti-hypertensive agents, antimicrobial factors, food intake modifiers
and immune regulatory factors [8,9]. Interestingly, many bioactive peptides are inactive within their parent milk proteins,
and upon release during digestion or food processing, they may act as regulatory compounds
with hormone-like activity [10,11]. Additionally, there are increasing studies showing that bioactive milk peptides
can be absorbed intact from the intestinal lumen into the blood circulation - these
may thus serve as novel functional food ingredients or pharmaceutical agents [12-15].

Using a proprietary enzyme digestion and buffer isolation method, we purified a group
of peptides from the whey fraction of regular cow's milk and screened against a panel
of kinases. In particular, this milk peptide mixture shows inhibitory effects against
EGFR, VEGFR2, and IR. EGFR is often overexpressed in non-small cell lung cancer (NSCLC)
and a variety of common solid tumors. EGFR signaling is generally associated with
cancer invasion, metastasis, chemotherapy resistance, and poor prognosis [16,17]. It has also been reported that inhibition of EGFR may lead to apoptosis in certain
cancer cell lines [18]. Gefitinib (Iressa®, AstraZeneca plc, London, UK) and Erlotinib (Tarceva®, Genentech, Inc., San Francisco, CA) are examples of anti-cancer drugs targeting EGFR-TK. Interestingly, MP mixture is
also able to cause significant cell death in HT29 colon cancer cells, whereas neither
commercially available hydrolyzed whey proteins nor whole milk proteins exhibited
the same property. This may be because most commercial hydrolyzed whey proteins contained
too small amount of bioactive peptides or the process of spray drying deactivated
the activity.

This novel MP mixture also inhibits insulin receptor signaling. Interestingly, mutations
in daf-2, a gene that encodes an insulin-like receptor in C. elegans worm, have been shown to double the lifespan of the worms [19]. The gene is known to regulate reproductive development, ageing, resistance to oxidative
stress, thermotolerance, resistance to hypoxia and also resistance to bacterial pathogens
[20]. Therefore, we next tested this unique MP mixture effect on the lifespan of C. elegans worms. Statistical analysis of the result suggested that N2 worms fed with agar containing
20 μg/ml concentration of milk peptides increased the median lifespan by 15.4% (p
= 0.014). Based on these data, we hypothesize that this milk peptide mixture may be
a novel supplement ingredient for anti-aging and cancer preventive regimen.

Since MP has multi-kinase inhibitory activity in the micromolar range, we first conducted
toxicology studies to measure the safety of these peptides. An acute and a 28-day
sub-acute toxicology study showed no apparent adverse effects in rodents following
oral administration of the peptides. Encouraged by the results, we conducted an 80
subject, double blind, placebo controlled study in healthy volunteers. The main purpose
was to assess the safety of the milk peptides at three escalating dosages.

The clinical analyses showed that the MP mixture did not cause significant side effects
in healthy human subjects. All blood and hormonal markers remained in normal ranges.
There was a mean trend toward improving insulin sensitivity as assessed using HOMAIR that warrants additional study. This could potentially have beneficial effects for
individuals with insulin resistance. However, these changes were not significant due
to the large variability in responses observed. Additional study evaluating the effects
of these milk peptides on glucose tolerance in individuals with and without insulin
resistance would help elucidate this potential effect.

Another encouraging trend was the lowering of neutrophil to lymphocyte ratio (NLR).
The neutrophil-lymphocyte ratio is a general marker of immunity and inflammatory status.
Research has shown that elevated NLR to be a prognostic indicator of poor chemotherapy
outcomes in advanced colorectal cancer and poor survival after colorectal liver metastases
[21-23]. The potential impact of MP supplementation on lowering NLR could be used as a clinical
nutritional intervention for these categories of cancer patients. In addition, these
peptides have been shown to have in vitro anti-EGFR and VEGFR2 activity. EGFR is commonly elevated in many advanced cancers,
and VEGFR2 is considered one of the key regulators of tumor-induced angiogenesis [24,25]. Together, this novel milk peptide mixture's anti-cancer and inflammatory reducing
properties may be a safe, effective supplement to help cancer patients undergoing
chemotherapy to obtain better clinical outcome.

In summary, preliminary experiments suggest that there may be some potentially beneficial
applications of this novel MP mixture and that six weeks of human consumption appears
to be safe. The limitation of this first clinical study is that the human subjects
were healthy individuals. Additional clinical studies are currently underway to further
elucidate the milk peptides' impact on the progression of cancer and quality of life
in cancer patients.

Competing interests

Ambryx Biotechnology, Inc. (Riverside, CA) provided funding for the clinical trial through an unrestricted grant to Baylor
University when the Principal Investigator and the Exercise & Sport Nutrition Lab
were affiliated with that institution. RBK, MI, MK, GH, and CR independently collected,
analyzed, and interpreted the results from the clinical trial and have no financial
interests concerning the outcome of this investigation. HC, OM and MT are research
scientists affiliated with Ambryx Biotechnology.

Authors' contributions

RBK served as Principal Investigator and contributed to the design of the study, statistical
analysis, manuscript preparation, and procurement of external funding. MI served as
the study coordinator, oversaw all testing, and assisted in data analysis. MC and
GH assisted in data collection and performed whole blood, serum, and hormonal assays.
CR served as the lab coordinator and supervised all data collection and assisted with
data analysis. HC, OM and MT supervised the conduct of basic research experiments
and consulted on the design of the clinical trial. All authors read and approved the
manuscript.

Acknowledgements

We would like to thank the individuals who participated in this study as well as all
of the students and administrative support staff's at Baylor University and Texas
A&M University who assisted in conducting the clinical trial. We would also like to
thank Dr. Ron Wilson for providing medical supervision.